A heat-stable endoribonuclease isolated from chicken liver has been purified to homogeneity as evidenced by the presence of a single protein upon polyarcrylamide gel electrophoresis. The enzyme can, in limited digests of 5 S rRNA and 5.8 S rRNA, distinguish between cytidylic and uridylic acids bonds at a ratio of 61:1 and, therefore, may be useful in RNA sequence analysis. The means by which the enzyme hydrolyzes substrate is unusual in that kinetic data do not support a simple formation and breakdown of an enzyme-substrate complex. Rather, the existence of a second complex, consisting of 2 mol of substrate and one of enzyme, derived from the initial enzyme-substrate complex, is postulated. In common with the other endonucleases, enzyme activity is inhibited by free poly(A) or tracts of the polypurine present at the 3'-terminus of RNA. Reversal of inhibition and restoration of activity may be achieved by the addition of low concentrations of spermidine to reaction mixtures.